A three-phase program for the detection, isolation and characterization of congenital enzyme defects of carbohydrate metabolism is proposed. The investigation is a continuation of studies on glucosephosphate isomerase (GPI) and triosephosphate isomerase (TPI) deficiency diseases, but will be expanded to examine pyruvate kinase (PK) deficiency. PK-deficiency is the second most common and GPI-deficiency the third most common erythroenzymopathies leading to chronic hemolytic anemias. TPI deficiency, while rare, is the most severe of all glycolytic enzyme defects, resulting in neuromuscular and mental retardation. In Phase I genetic variants of the enzyme are identified on the basis of their catalytic and electrophoretic properties. A variety of genetic variants of GPI have been identified with altered catalytic, chemical or physical properties and are currently under investigation. The enzymes are purified to homogeneity by affinity chromatography and the allozymes resolved isoelectric focusing. In Phase II the catalytic, regulatory, immunological and stability properties of the enzymes are quantified. These studies also assess the "effective catalytic capability" of the normal and mutant enzymes undersimulated physiological conditions. Phase III is designed to elucidate the structural basis of the mutations by utilizing sensitive microsequencing capabilities which permit peptide isolation and sequence determination at the picomole level. Completion of Phase III studies on the isozymes of TPI should establish the molecular basis for the isozymes and reveal whether TPI-deficiency is due to a structural gene mutation affecting the A isozyme or a mutation preventing the conversion of TPI-B to TPI-A. These studies will not only provide a complete understanding of the molecular basis of these enzyme deficiencies, but should also provide a better understanding of the more subtle regulatory interactions of carbohydrate metabolism.